Non patent literature 1 indicates that the three-dimensional culture method is available for reproducing the functions of living tissues more accurately than the two-dimensional culture method. Non patent literature 1 also indicates that the three-dimensional culture method is one of methods useful for efficiently differentiating pluripotent stem cells and iPS cells. Attempts are being made to rebuild and complement lost functions by returning three-dimensionally cultured artificial tissues into a body using such a technique. Moreover, other attempts are being made to apply such a technique to regenerative medicine for assistance of damaged tissue repair. Further attempts are being made to use such a technique for testing toxicity of pharmaceutical agents. In particular, large-scale production of cell aggregates that are uniform in size and shape is required for regenerative medicine and its research. In addition, a method with higher convenience and lower cost than existing methods will become necessary.
[Significance of Producing Uniform Sized and Shaped Cell Aggregates]
In the case of liver cells, there is a problem that in vivo functions cannot be reproduced in vitro. One example of this problem is that the drug metabolism function inherent in liver cells is deteriorated when the liver cells are two-dimensionally cultured. One of means for addressing this problem is a method for forming cell aggregates as disclosed in Non-patent literature 2. Non patent literature 2 indicates that the function of the cell aggregates produced by this method is dramatically improved from those of the two-dimensionally cultured cells.
When embryonic stem cells or induced pluripotent stem cells are differentiated into target cells in vitro, cell aggregates called embryoid bodies is formed, followed by the initiation of the program of development and differentiation, to thereby differentiate embryonic stem cells OF induced pluripotent stem cells into target cells. Non patent literature 3 reports that size of cell aggregates influence the directions of differentiation.
[Regarding Technique for Producing a Large Number of Uniform Size and Shape Cell Aggregates]
Non patent literature 4 discloses a culture method called the hanging drop method in which culture is performed in droplets. Non patent literature 4 further discloses a U or V bottomed low adherence plate. Non patent literature 4 also discloses a large-scale culture method using bioreactors. The hanging drop method, U bottomed plate and the like are suitable for producing cell aggregates that are uniform in size and shape. On the other hand, the hanging drop method, U bottomed plate and the like are not suitable for large-scale culture because they allow only one cell aggregate to be produced in one well. Although the hanging drop method is widely applied to research, it is not suitable for large-scale culture for producing more than several hundred or several thousand order of cell aggregates at a time.
Non patent literature 4 discloses a method using a low adherence container. Non patent literature 4 also discloses a development of a method using roller bottle. Further disclosed is a development of a method for immobilizing cells in a gel or beads so as to carry out suspension culture. Although these methods enable several thousand cell aggregates to be produced with a high density, there is a problem that the produced cell aggregates are heterogeneous. Roller bottles in low rotation that are capable of large-scale production of comparatively homogeneous cell aggregates are being developed. However, these methods require a large-scale apparatus accompanied with complicated operation. Further, even though the rotation speed of roller bottles or the like is controlled precisely, any adjacent cells and cell aggregates may be irregularly associated with each other to form another cell aggregates in a solution. It is thus difficult to produce uniform size cell aggregates.
For example, patent literature 1 discloses an example of a method for producing a group of cell aggregates in a large number and with convenience. In this group, morphological features such as size and shape of the cell aggregates and properties outside cells are homogeneous. In the method disclosed in patent literature 1, a culture solution containing cells is poured into a hollow part of a structural member, wherein at least one lower end of the hollow part is opened. At this time, a portion of the culture solution is made to project downward from the open end. In this method, the cells are cultured in the projecting portion of the culture solution. This method has a problem for pouring the culture solution into the hollow part when an upper end of the hollow part is closed. Another problem of this method is that a process for forming the projecting portion of the culture solution is complicated. Moreover, a problem in this process is that it is necessary to accurately adjust an amount of pouring. Although the pouring (suction) of the culture solution can be efficiently carried out when both ends of the hollow part are open, there is another problem in this method. The problem is that the upper end needs to be closed or a mechanism for maintaining a suction pressure needs to be further included in order to maintain a state of the culture solution that has been poured into. New limitations are imposed on this manner since an amount of the culture solution that can be involved in metabolism of the cells is regulated by a volume of the hollow part. One of the new limitations is that a cell concentration and a culture period are limited by the metabolic activity of a cell for use. The available size of cell aggregates and types of cells are also limited by this new limitation. Patent Literature 2 discloses a suspension plate that allows easier pouring of a culture solution than that in the technique disclosed in Patent literature 1. Patent literature 2 discloses a structure in which a culture solution is communicated from a first surface with a second surface. However, with the manner disclosed in patent literature 2, the number of pouring operations is increased in proportion to the number of cell aggregates to be created. Accordingly, the problem of complicated operations still remains.